The goal of this work is to produce a platform for studying bacterial group behavior under controlled environmental conditions, allowing fundamental questions to be investigated regarding the onset and maintenance of states corresponding to virulence and enhanced antibiotic and immune resistance. In these studies, a novel approach based on multiphoton lithography (MPL) will be developed for microfabricating defined, arbitrary topographies using biological building blocks (proteins) which will be investigated as a means to produce microcavities capable of supporting cell aggregration, growth, and group behavior, focusing on quorum sensing and biofilm deposition by the opportunistic environmental bacterium, Pseudomonas aeruginosa, as a model system. This bacterium has relevance to a number of human diseases, including persistant infections in cystic fibrosis individuals. Toward these ends, the proposed studies will advance the underlying MPL technology, focusing on characterization of material and microcavity properties, and will apply this state-of-the-art technology to an evaluation of P. aeruginosa behavior in protein-based microcavities. This project represents collaborative studies between Prof. Jason Shear's research group (Chemistry/Biochemistry Department), and that of Prof. Marvin Whiteley (Microbiology Department) at UT Austin. It is anticipated that the data generated will provide important information for future studies aimed at identifying novel therapeutics targeting bacterial communication systems. Although we are proposing P. aeruginosa as the test bacterium for these studies, they could easily be extended to other bacterial species and even to more complex multi-species communities. The Specific Aims are: (1) To Optimize a flexible platform for fabricating 3D bacterial microcavities having tunable mass-transport to the outside environment;and (2) To assess the utility of 3D protein-based microcavities for studying bacterial group behavior. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to develop a platform for studying bacterial group behavior under controlled environmental conditions, allowing fundamental questions to be investigated regarding the onset and maintenance of states corresponding to virulence and enhanced antibiotic and immune resistance. In these studies, a novel approach for microfabricating defined, arbitrary topographies using biological building blocks will be investigated as a means to produce microcavities capable of supporting cell aggregration, growth, and group behavior. It is anticipated that the data generated will provide important information for studies aimed at identifying novel therapeutics targeting bacterial communication systems.